Gluconacetobacter diazotrophicus

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Classification

Bacteria; Proteobacteria; Alphaproteobacteria; Rhodospirillales; 'Acetobacteraceae'; Gluconacetobacter

Species

NCBI: [1]

Gluconacetobacter diazotrophicus

Description

Figure 1 : Electron micrograph of a G. diazotrophicus cell. Peritrichous flagella are visible. The dimensions of the cell are 0.7-0.9 μm x 2 μm. Photo taken by M. Gillis, K. Kersters, B. Hoste et al. (1989)[2]

Gluconacetobacter diazotrophicus is a symbiotic, plant-growth promoting bacteria. It was isolated from the roots and stems of Brazilian sugarcane plants in 1988 [1]. Upon discovery, the bacterium was named Saccharobacter nitrocaptans. Due to its acetic acid production and similarity to previously classified bacteria, however, it was later renamed to Acetobacter diazotrophicus. Completion of 16S ribosomal RNA analysis led to a reclassification to its current designation and taxonomy [1,2,3].

G. diazotrophicus is a Gram-negative, nonspore forming and nitrogen fixing obligate aerobe [2]. The temperature and pH growth optimums are 30°C and 5.5 respectively. The bacterium is acid-tolerant and can also both grow and fix nitrogen at pH of 3.0 and below [1,2]. Additionally, G. diazotrophicus grows optimally at a sucrose concentration of 10%, as found in its natural host, but is capable of growth at up to 30% sucrose under laboratory conditions. The bacteria has been shown to grow abundantly on other carbon substrates like D-galactose, D-fructose, and D-mannose [1].

Unlike many other bacteria that engage in symbiosis with plants, G. diazotrophicus is an endophyte and does not stimulate the production of nodules [1]. Without a host plant, the bacteria will not survive in the soil for more than two days [4]. Most host plants of G. diazotrophicus contain relatively high levels of sucrose, similar to the sugarcane on which it was discovered [5].

Significance

Figure 2. Corn and wheat, two monocot crops of agricultural significance, pictured side by side. Photo retrieved from: [9]

The ability of G. diazotrophicus to fix nitrogen and effectively promote the growth of its host plant opens the possibility for agricultural applications. Additionally, G. diazotrophicus has many other attractive characteristics. The bacteria is of monocot origin, less plant specific than other symbiotic nitrogen fixing bacteria, and does not require nodule structures for growth and nitrogen fixation [6]. Given these factors, G. diazotrophicus could be a less costly and more environmentally friendly alternative to nitrogen fertilizers that the agricultural industry currently relies on heavily [6,7]. The bacteria could be adapted to colonize other monocot plants if sucrose levels were not a limiting factor. Monocot staples in agriculture include corn, wheat, and rice. These crops account for approximately 70% of the total world crop production [8]. If nitrogen fertilizers could be supplemented or replaced by G. diazotrophicus colonization in these crops, that could lead to more sustainable and less environmentally damaging agricultural practices on a large scale [6].

Genome Structure

G. diazotrophicus contains the Pal5 genome consisting of one circular chromosome (3,944,163 bp) and a G-C content of 66.19%. Additionally there are two plasmids present, pGDO1 (38,818bp) and pGDO2 (16,610bp). The genome was found to contain 3864 coding sequences. There were 1077 potential proteins sequenced in the genome, with 583 being identified to the metabolic pathways within G. diazotrophicus. Studies have shown that expression is dependent on the crop present (such as sugar cane) and the levels of nitrogen present in the environment. Genes were found to augment resistance to acetic acid, which allows G. diazotrophicus to fix nitrogen in very acidic environments. [10]

Cell Structure, Metabolism and Life Cycle

Cell Structure

The bacterium’s cells are shaped like straight rods with rounded ends and motility is provided by 1-3 lateral or peritrichous flagella. Cellular dimensions are approximately 0.7-0.9 μm x 2 μm [2]. When viewed under a microscope, cells are single, paired, or chainlike in structure.

Nitrogen Fixation

G. diazotrophicus reduces atmospheric nitrogen to ammonium by a molybdenum (Mo)-dependent nitrogenase [11]. Mo-dependent nitrogenase contains Fe protein and MoFe protein [12]. The Fe protein is a homodimer that has a binding site for MgATP in each subunit. When the Fe protein is complexed with MoFe and two MgATP molecules, the MgATP is hydrolyzed to MgADP, and the MoFe protein binds the substrate for reduction [12]. The bacteria’s lack of a nitrate reductase protein suggested that nitrogen fixation would not be inhibited by feedback from nitrogen assimilation, but nitrate still may inhibit the process [3,13]. Low levels of fertilizer do not inhibit nitrogen fixation, so G. diazotrophicus may be a useful supplement to ammonium based-fertilizers [11,14].

Figure 3. Molybdenum-dependent nitrogenase. G. diazotrophicus uses a Mo-dependent nitrogenase for nitrogen fixation. The Fe protein binds and hydrolyzes ATP, and the MoFe protein binds the substrate [32].

Carbon Metabolism

G. diazotrophicus is a chemoorganoheterotroph. Glycolysis and the pentose phosphate pathway are found in G. diazotrophicus [15]. Its carbon sources include d-galactose, d-arabinose, d-fructose, and d-mannose [3]. The main pathway for glucose oxidation occurs in the periplasm. Pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH) oxidizes glucose to gluconate extracellularly [16, 17]. The enzyme is produced to meet the energy requirements during nitrogen fixation [17]. G. diazotrophicus also uses nicotinamide adenine dinucleotide-linked glucose dehydrogenase (NAD-GDH) for intracellular glucose oxidation [18]. Levansucrase, a fructosyltransferase exoenzyme, helps G. diazotrophicus to survive in high sucrose environments by aiding in sucrose transport [19]. The enzyme acts extracellularly to hydrolyze sucrose to fructooligosaccharides and levan [19, 20]. The levan produced by the enzyme is also critical for biofilm formation by the bacteria [21, 22].

Life Cycle

G. diazotrophicus can live and reproduce intracellularly or extracellularly within plants. Sugarcane produces anionic glycoproteins capable of binding G. diazotrophicus as a mechanism of selection for the endophyte [23]. The bacteria are more likely to enter the plant at the root cap on root tips where there are loose cells. Entry through the stem occurs at openings where young plants have diverged. In leaves, G. diazotrophicus can enter a wounded stomata [24]. Vector transmission by the sap eating pink sugarcane mealybug, Saccharococcus sacchari, is also possible [25]. Production of bacterial hydrolytic enzymes assists in colonization [24,26]

Ecology and Symbiosis

Figure 4. Damage to X. albilineans structure. Transmission electron micrograph of X. albilineans when treated with gluconacin [31].

G. diazotrophicus was initially isolated from sugarcane in Brazil but later found to be associated with sugarcane in Philippines, Japan, Argentina, Cuba, Mexico, Australia, Mauritius, and India [15]. It is an endophyte that can colonize intracellular or intercellular spaces in sugarcane roots, leaves, and stems. In other crops like carrots, radish, beetroot, coffee, and rice, G. diazotrophicus has been isolated from the rhizosphere [15].

Plant Growth Promotion

Carbon sources from plants benefit the bacteria, but there are numerous ways G. diazotrophicus supports plant growth. Nitrogen fixation by G. diazotrophicus produces about half of the required nitrogen for crops [27,28]. The bacteria also promotes plant growth through phytohormone production. Production of indole-3-acetic acid and gibberellins promotes growth of the root systems and shoot systems in plants [29]. G. diazotrophicus provides nutrients to plants by solubilization of phosphorus and zinc [6]. Outside of growth promotion, G. diazotrophicus protects sugarcane from Xanthomonas albilineans, the causative agent of leaf scald, which results in wilting, necrosis, and plant death [30]. Production of a Linocin M-18 like bacteriocin called gluconacin prevents leaf scald by lysing X. albilineans [31]. The bacteriocin is highly conserved in the Actetobaceterceae. The bacteria’s antagonistic capabilities make it a candidate for biocontrol in agriculture. Potential uses of gluconacin include application of teh bacteriocin of gluconacin to plants and tools to prevent infection by X. albineans. Engineering of transgenic plants capable of producing gluconacin could further prevent leaf scald in crops [30].

Human Pathogenesis

G. diazotrophicus shares low 16s rRNA similarity with the two human pathogens within the Acetobacteraceae family. It has no history of infecting humans [15].

References

[1] V. A. Cavalcante and J. Dobereiner. “A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane.” Plant and Soil. 1988. Volume 108. No. 1. p. 23–31.

[2] M. Gillis, K. Kersters, B. Hoste et al. “Acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane.” International Journal of Systematic Bacteriology. 1989. Volume 39. No. 3. p. 361–364.

[3] Y. Yamada, K.-I. Hoshino, and T. Ishikawa. “The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus gluconoacetobacter to the generic level.” Bioscience, Biotechnology and Biochemistry. 1997. Volume 61. No. 8. p. 1244–1251.

[4] M. A. Paula, V. M. Reis, and J. Döbereiner. “Interactions of Glomus clarum with Acetobacter diazotrophicus in infection of sweet potato (Ipomoea batatas), sugarcane (Saccharum spp.), and sweet sorghum (Sorghum vulgare).” Biology and Fertility of Soils. 1991. Volume 11. No. 2, p. 111–115.

[5] P. J. Riggs, M. K. Chelius, A. L. Iniguez, S. M. Kaeppler, and E. W. Triplett. “Enhanced maize productivity by inoculation with diazotrophic bacteria.” Australian Journal of Plant Physiology. 2001. Volume 28. No. 9. p. 829–836.

[6] N. Eskin, K. Vessey, L. Tian. "Research Progress and Perspectives of Nitrogen Fixing Bacterium, Gluconacetobacter diazotrophicus, in Monocot Plants." International Journal of Agronomy. 2014. Volume 2014. Article ID 208383. 13 pages.

[7] M. B. Peoples, D. F. Herridge, and J. K. Ladha. “Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production?” Plant and Soil. 1995. Volume 174. No. 1-2. p. 3–28.

[8] Van Vu, T., Sung, Y.W., Kim, J. et al. Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants. Rice. 2019. Volume 12. Article number 95.

[9] Lakeviewgrains2323243243. (2017, December 6). Wheat Technology Races to Catch Soybean and Corn. Lakeview Grains.

[10] N. Eskin, K. Vessey, L. Tian, Research Progress and Perspectives of Nitrogen Fixing Bacterium, Gluconacetobacter diazotrophicus, in Monocot Plants. International Journal of Agronomy. 2014. Volume 2014. Article ID 208383. 13 pages. https://doi.org/10.1155/2014/208383

[11] Fisher, K., & Newton, W. E. (2005). Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium. Biochimica Et Biophysica Acta (BBA) - Proteins and Proteomics. 2005. Volume 1750. No. 2. p. 154-165. ]https://doi.org/10.1016/j.bbapap.2005.04.010

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[13] Trinchant, J.-C., & Rigaud, J. Nitrite and nitric oxide as inhibitors of nitrogenase from soybean bacteroids. Applied and Environmental Microbiology. 1982. Volume 44. No. 6. p. 1385-1388. https://doi.org/10.1128/aem.44.6.1385-1388.1982

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[15] Saravanan, V. S., Madhaiyan, M., Osborne, J., Thangaraju, M., & Sa, T. M. Ecological occurrence of Gluconacetobacter diazotrophicus and nitrogen-fixing Acetobacteraceae members: Their possible role in plant growth promotion. Microbial Ecology. 2007. Volume 55. No. 1. p. 130–140. https://doi.org/10.1007/s00248-007-9258-6

[16] Attwood, M. M., van Dijken, J. P., & Pronk, J. T. Glucose metabolism and gluconic acid production by Acetobacter diazotrophicus. Journal of Fermentation and Bioengineering. 1991. Volume 72. No. 2. p. 101–105. https://doi.org/10.1016/0922-338x(91)90317-a

[17] Galar, M. L., & Boiardi, J. L. Evidence for a membrane-bound pyrroloquinoline quinone-linked glucose dehydrogenase in Acetobacter diazotrophicus. Applied Microbiology and Biotechnology. 1995. Volume 43. No. 4. p. 713–716. https://doi.org/10.1007/bf00164778

[18] Luna, M. F., Bernardelli, C. E., Galar, M. L., & Boiardi, J. L. Glucose metabolism in batch and continuous cultures of Gluconacetobacter diazotrophicus PAL 3. Current Microbiology. 2006. Volume. 52. No. 3. p. 163–168. https://doi.org/10.1007/s00284-005-4563-0

[19] Martínez-Fleites, C., Ortíz-Lombardía, M., Pons, T., Tarbouriech, N., Taylor, E. J., Arrieta, J. G., Hernández, L., & Davies, G. J. Crystal structure of levansucrase from the gram-negative bacterium Gluconacetobacter diazotrophicus. Biochemical Journal. 2005. Volume 390. No. 1. p. 19–27. https://doi.org/10.1042/bj20050324

[20] Hernandez, L., Arrieta, J., Menendez, C., Vazquez, R., Coego, A., Suarez, V., Selman, G., Petit-Glatron, M. F., & Chambert, R. Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochemical Journal. 1995. Volume 309. No. 1. p. 113–118. https://doi.org/10.1042/bj3090113

[21] Velázquez-Hernández, M. L., Baizabal-Aguirre, V. M., Cruz-Vázquez, F., Trejo-Contreras, M. J., Fuentes-Ramírez, L. E., Bravo-Patiño, A., Cajero-Juárez, M., Chávez-Moctezuma, M. P., & Valdez-Alarcón, J. J. Gluconacetobacter diazotrophicus levansucrase is involved in tolerance to NaCl, sucrose and desiccation, and in biofilm formation. Archives of Microbiology. 2010. Volume 193 No. 2. p. 137–149. https://doi.org/10.1007/s00203-010-0651-z

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[23] Estrella Legaz Marı́a, Pedrosa, M. M., Armas, R. de, Rodrı́guez Carlos W., Rı́os Vivian de, & Vicente, C. Separation of soluble glycoproteins from sugarcane juice by capillary electrophoresis. Analytica Chimica Acta. 1998. Volume 372. No. 1-2. p. 201–208. https://doi.org/10.1016/s0003-2670(98)00331-6

[24] James, E. K., Olivares, F. L., de Oliveira, A. L. M., dos Reis, F. B., da Silva, L. G., & Reis, V. M. Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions. Journal of Experimental Botany. 2001. Volume 52. No. 357. p. 747–760. https://doi.org/10.1093/jexbot/52.357.747

[25] James, E. K., & Olivares, F. L. Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs. Critical Reviews in Plant Sciences. 1998. Volume 17. No. 1. p. 77–119. https://doi.org/10.1080/07352689891304195

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[27] Boddey, R. M., Polidoro, J. C., Resende, A. S., Alves, B. J., & Urquiaga, S. Use of The15n natural abundance technique for the quantification of the contribution of N2 fixation to sugar cane and other grasses. Functional Plant Biology. 2001. Volume 28. No. 9. p. 889. https://doi.org/10.1071/pp01058

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[29] Piccoli, P., Travaglia, C., Cohen, A., Sosa, L., Cornejo, P., Masuelli, R., & Bottini, R. An endophytic bacterium isolated from roots of the halophyte Prosopis strombulifera produces ABA, IAA, Gibberellins A1 and A3 and jasmonic acid in chemically-defined culture medium. Plant Growth Regulation. 2010. Volume 64. No. 2. p. 207–210. https://doi.org/10.1007/s10725-010-9536-z

[30] Oliveira, M. M., Ramos, E. T. A., Drechsel, M. M., Vidal, M. S., Schwab, S., & Baldani, J. I. Gluconacin from Gluconacetobacter diazotrophicus PAL5 is an active bacteriocin against phytopathogenic and beneficial sugarcane bacteria. Journal of Applied Microbiology. 2018. Volume 125. No. 6. p. 1812–1826. https://doi.org/10.1111/jam.14074

[31] Almeida Ramos, E. T., Meneses, C. H., Vidal, M. S., & Baldani, J. I. Characterisation and action mode of gluconacin, a bacteriocin with antimicrobial activity against Xanthomonas albilineans. Annals of Applied Biology. 2021. Volume 180. No. 1. p. 163–175. https://doi.org/10.1111/aab.12721

[32] Jimenez-Vicente, E., Yang, Z.-Y., Ray, W. K., Echavarri-Erasun, C., Cash, V. L., Rubio, L. M., Seefeldt, L. C., & Dean, D. R. Sequential and differential interaction of Assembly factors during nitrogenase MOFE protein maturation. Journal of Biological Chemistry. 2018. Volume 293. No. 25. p. 9812–9823. https://doi.org/10.1074/jbc.ra118.002994

Authors

Page authored by Isaac Coker, Kyra Colston, and Danielle DeCesaris, students of Prof. Jay Lennon at Indiana University.